Monitoring minimal residual disease in peripheral blood in B-lineage acute lymphoblastic leukaemia

The use of peripheral blood rather than marrow has potential advantages for monitoring minimal residual disease during the treatment of leukaemia. To determine the feasibility of using blood, we used a sensitive polymerase chain reaction method to quantify leukaemia in the blood and marrow in 35 paired samples from 15 children during induction treatment. Leukaemic cells in the blood ranged from 1.1 × 10⁻² to < 9.4 × 10⁻⁷ leukaemic cells/total cells, corresponding to 1.3 × 10⁷ to < 2 × 10³ leukaemic cells/l. In 15 paired samples, leukaemia could be quantified in both tissues and in 20 paired samples, leukaemia was not detected in one or both tissues so that only upper level limits could be set. In the former 15 pairs, the level of leukaemia in peripheral blood was directly proportional to that in marrow but was a mean of 11.7-fold lower. Leukaemia in blood was detected in 10/12 pairs in which the level in marrow was > 10⁻⁴, but in only two of 13 pairs in which the level in marrow was < 10⁻⁵. Patients studied at multiple time-points showed parallel declines in the number of leukaemic cells in both tissues. The results showed that leukaemia could be monitored in peripheral blood during induction therapy, and quantitative considerations based on the results suggest that monitoring of blood during post-induction therapy may be of value in detecting molecular relapse.     

INK4A/ARF deletions are acquired at relapse in childhood acute lymphoblastic leukaemia: a paired study on 25 patients using real-time polymerase chain reaction

Current risk-adjusted intensive therapies for childhood acute lymphoblastic leukaemia (ALL) are expected to result in an event-free survival of greater than 75%. In sharp contrast, relapsed paediatric ALL is a difficult disease to treat. In this study, 25 paediatric patients with ALL were analysed at diagnosis and relapse for their p16 (exon 2) status using the most accurate method of detection, real-time polymerase chain reaction (PCR). The median time to relapse for the group was 27 months. At diagnosis, the incidence of p16 homozygous and hemizygous deletion in this group was 32% and 20% respectively. The incidence of homozygous p16 deletion at relapse was 64%. A large number of patients, eight of 16 (50%), developed p16 homozygous deletion at relapse. Of those eight patients, four were hemizygous and four were germline at diagnosis. At diagnosis, those patients with a homozygous or hemizygous p16 deletion relapsed sooner than those germline for p16. We have shown that p16 alterations are frequently present in relapsed lymphoblastic leukaemia in children.     

Interleukin 8 in serum in granulocytopenic patients with infections

Serum levels of interleukin 8 (IL-8) were examined in eight patients with acute myeloid leukaemia during 16 courses of chemotherapy. The patients experienced 14 episodes of fever which occurred in periods with granulocyte counts <0·5 × 10⁹/l. Febrile episodes were classified as bacteriologically defined infection ( n = 6), clinically defined infection ( n = 2), and unexplained fever ( n = 6). IL-8 was detected in 18/25 (72%), 2/3 (67%) and 3/7 (43%) of the serum samples in the respective groups. In contrast, IL-8 was detected in 22/90 (24%) of the samples taken when no fever was present ( P <0·00003 versus bacteriologically defined infection). The median concentration of IL-8 in samples taken during febrile episodes was 194 ng/ml (range 0–6358 ng/ml) and 0 (range 0–5392 ng/ml) on days without fever (not significant). In three patients with infections caused by, respectively, Streptococcus sanguis, Acinetobacter calcoanitratus and Candida albicans , IL-8 rose to a peak levels and declined during recovery. We conclude that IL-I is released systemically during infections with gram-positive and gram-negative bacteria and Candida albicans in patients with acute myeloid leukaemia and peripheral granulocytopenia due to chemotherapy. However, IL-8 can also be detected when no sign of infection is present.     

Cytotoxicity of 2-chlorodeoxyadenosine and arabinosylcytosine in leukaemic lymphoblasts from paediatric patients: significance of cellular nucleoside transporter content

2-chlorodeoxyadenosine (2-CdA) and arabinosylcytosine (araC) are nucleoside drugs that are used to treat various leukaemias, although 2-CdA has not been tested extensively in children with acute lymphoblastic leukaemia (ALL). Nucleoside cytotoxicity depends on the conversion of these agents to 5'-phosphate derivatives, following drug entry into cells via nucleoside transport (NT) processes. This study compared es nucleoside transporter content, determined using a flow cytometric assay with SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] fluorescein, and cytotoxicities of 2-CdA and araC in fresh lymphoblasts from previously untreated paediatric ALL patients and the human T-lymphoblast cell line, CCRF-CEM. Lymphoblast samples from individual patients ranged widely in sensitivity to both 2-CdA (IC50, 6 nmol/l to > 5 micromol/l; mean = 418 nmol/l; n = 8) and araC (IC50, 59 nmol/l to > 5 micromol/l; mean = 1050 nmol/l; n = 7), although IC50 values for the two drugs were correlated (r = 0.78, P = 0.032, n = 7). Cellular es nucleoside transporter content varied more than 35-fold among samples from 10 patients. The correlation between es nucleoside transporter content and drug sensitivity was statistically significant for araC (r = -0.93, P = 0.023, n = 5), but not for 2-CdA (r = -0.57, P = 0.23, n = 6). Exposure of CCRF-CEM cells to araC resulted in a substantial araC concentration-dependent increase in the relative survival of es transporter-deficient cells, whereas the increase was slight following exposure to 2-CdA. We conclude that, in ALL lymphoblasts, es nucleoside transporter content is a determinant of araC sensitivity and that a deficiency in NT may impart resistance to araC.     

Understanding and harnessing the graft-versus-leukaemia effect

The graft-versus-leukaemia (GVL) effect is a central component of the stem cell allograft's ability to cure haematological malignancies. The GVL effect is mediated by donor-derived natural killer cells and T lymphocytes, which have distinct mechanisms of recognizing and targeting the recipient's malignant cells. After transplantation the cytokine milieu is favourable to the early establishment of a GVL effect, but the need to prevent graft-versus-host disease limits the full potential of this process. Clinical studies have identified some critical components of the transplant preparation, donor selection, stem cell source (peripheral blood versus bone marrow) and post-transplant management that can be manipulated to optimize the GVL effect. However, further developments focusing on the selective depletion of unwanted alloreactivity with preservation of GVL effects, and the use of vaccines or the adoptive transfer of leukaemia-specific lymphocytes, will be required to enhance the GVL effect to reliably eradicate more resistant leukaemias.     

All-trans retinoic acid-induced downregulation of annexin II expression in myeloid leukaemia cell lines is not confined to acute promyelocytic leukaemia

Most acute promyelocytic leukaemia (APL) patients suffer from disordered haemostasis. APL can be treated successfully in most instances by all-trans retinoic acid (ATRA) therapy, which induces endpoint maturation of the leukaemic promyelocytes with the characteristic t(15;17). Annexin II (AnII), a profibrinolytic protein, has been implicated in the bleeding manifestation seen in APL. Our group has shown previously that high levels of AnII are expressed on other acute myeloid leukaemia subtypes that are sometimes associated with disordered haemostasis, albeit less frequently than APL. This study examined the effects of ATRA on AnII expression and cell differentiation, on myeloid leukaemia cell lines to determine whether a regulatory influence on AnII may contribute to the return of haemostatic stability in APL following treatment. The results confirmed that AnII expression in the APL cell line (NB4) was significantly downregulated in response to ATRA (P < 0.01), with associated morphological and immunophenotypical evidence of myeloid differentiation. ATRA also downregulated AnII expression on other myeloid cell lines, albeit to a lesser extent than observed on NB4 cells. The results provide evidence that ATRA may resolve the hyperfibrinolysis in APL by downregulation of AnII expression.     

Prognostic impact of karyotypic findings in childhood acute lymphoblastic leukaemia: a Nordic series comparing two treatment periods. For the Nordic Society of Paediatric Haematology and Oncology (NOPHO) Leukaemia Cytogenetic Study Group

The prognostic impact of acquired chromosome abnormalities was evaluated in a population-based consecutive series of 768 children (< 15 years of age) with acute lymphoblastic leukaemia (ALL). The study cohort included all cases of cytogenetically abnormal childhood ALL diagnosed between 1986 and 1997 in the five Nordic countries (Denmark, Finland, Iceland, Norway and Sweden). The probability of event-free survival (pEFS) for the total cohort was 0. 72 +/- 0.02. When comparing the two treatment periods of July 1986 to December 1991 and January 1992 to December 1997, a better survival was seen for the latter time period (pEFS of 0.69 +/- 0.02 vs. 0.76 +/- 0.02, P = 0.05). Hypodiploidy with less than 45 chromosomes, t(9;22)(q34;q11) and 11q23 translocations were associated with a dismal outcome during the whole study period (pEFS of 0.57 +/- 0.12, 0.41 +/- 0.14 and 0.37 +/- 0.10 respectively). The poor prognostic influence of 11q23 rearrangements seemed to be restricted to infants and older children (> 10 years), who differed significantly from children aged 1-10 years in this regard (P < 0. 01). Patients with t(9;22)-positive ALL seemed to benefit from allogeneic bone marrow transplantation in first remission (P = 0.05). The pEFS for children with t(1;19)(q23;p13)-positive ALL was intermediate (0.63 +/- 0.17), with a tendency to a better outcome for patients with the unbalanced variant der(19)t(1;19). Hyperdiploid ALL patients, subdivided into moderate hyperdiploidy (47-51 chromosomes), massive hyperdiploidy (52-60 chromosomes) and cases in the tri-/tetraploid range (> 60 chromosomes) had the best outcome in the last treatment period (pEFS of 0.81 +/- 0.06, 0.80 +/- 0.04 and 0.88 +/- 0.07 respectively), unless t(1;19), t(8;14), t(9;22) or 11q23 translocations were present. In a multivariate analysis including white blood cell (WBC) count, immunophenotype, age, mediastinal mass, central nervous system involvement and leukaemia karyotype, only WBC and modal chromosome number were shown to be significant independent risk factors (P < 0.01).     

Development of extended multidrug resistance in HL60 promyelocytic leukaemia cells

Summary. In an attempt to mimic clinical conditions for the treatment of leukaemia, the HL60 promyelocytic cell line was treated for 18 h with low, clinically relevant, levels of the anthracycline epirubicin and the Vinca alkaloid vinblastine. The resulting drug-resistant sublines not only expressed P-glycoprotein and the MDR phenotype but were also cross-resistant to chlorambucil, methotrexate but were also cross-resistant to chlorambucil, methotrexate and cisplatinum, and had increased resistance to radiation. Development of resistance was associatted with an aberrant differentiation phenotype with decreased expression of myeloid antigens and expression of glycophorin A. an antigen normally associated with erythroid differentiation. The ability of HL60 cells to terminally differentiate in response to all- trans -retinoic acid (vitamin A acid) was lost in the sublines. These results suggest that either a single novel mechanism is responsible for multiple drug resistance or the initial response to drug treatment is the co-induction of multiple mechanisms. These cells and the method by which they were generated therefore provide a clinically relevant model for the study of the initial events in the development of not only multidrug resistance but also the extended multiple drug resistance usually encountered in the treatment of leukaemia.